The purpose of this investigation is to study an onco-developmental protein produced by tumor cells growing in cell culture. The synthesis, metabolism, molecular variants and physicochemical properties of rat alpha-fetoprotein (AFP) produced by mass and clonal cultures of cells derived from hepatocellular carcinomas will be studied. AFP produced by the cells in culture will be compared to AFP produced in vivo by normal fetal/newborn animals and by the hepatocellular carcinomas. Hepatocellular carcinoma-derived cells are grown using methods which have been modified to support the growth of epithelial cells. Mass and clonal cell lines have been established from Morris hepatoma 7777 and do produce AFP. The relationship of these cells to liver parenchymal cells will be established by cell morphology, albumin synthesis, and tyrosine aminotransferase activity. The synthesis, processing, excretion and molecular variants of AFP produced by the cultured cells will be characterized. Varying culture conditions, hormones, cyclic nucleotides and carcinogens on AFP production in vitro will be tested. Chromosome analysis of the cells will be performed using conventional and banding methods. AFP will be isolated by anti-rat AFP immunoadsorbent affinity chromatography. Concanavalin A-affinity molecular variants of AFP will be isolated by concanavalin A-affinity chromatography. Electrophoretic molecular variants of AFP will be isolated by preparative isoelectric focusing. The amino acids composition, peptide content, terminal amino acids, and carbohydrate content of the molecular variants of AFP produced by cells in culture will be determined by standard methods. Molecular size of AFP's will be determined by SDS-acrylamide gel electrophoresis and by gel filtration chromatography.